ABSTRACT Even as malaria control efforts have achieved great reductions in malaria burden over the last decade, vast numbers of asymptomatic infection exist and present an obstacle to malaria elimination. Most asymptomatic carriers harbor parasites that are detectable by PCR but missed by current rapid diagnostic tests and microscopy. These submicroscopic infections are one rationale for mass drug administration efforts. Yet the role of submicroscopic infections in sustaining transmission is unknown. In fact, the malaria parasites responsible for human to mosquito infection, gametocytes, may not achieve transmissible levels in the majority of submicroscopic infections. We propose that a better understanding of gametocyte-mediated transmission at low densities can lead to the design of better strategies to interrupt parasite transmission. Aim 1 of this proposal will use mosquito feeding assays to define who within the asymptomatic reservoir is infectious to mosquitoes, investigating the role of gametocyte density, sex ratio, and strain diversity in determining transmissibility. We will use direct skin feeding to measure human to mosquito transmission, considered a truer, more sensitive measure of human infectiousness than more commonly used membrane feeding assays. Aim 2 will determine the propensity for submicroscopic infections to persist and develop infective gametocytes over time. In these two aims, next generation sequencing techniques will be used to distinguish individual gametocyte strains to better understand how the composition of mixed gametocyte populations within individuals changes over time and affects transmission, and whether intensive control efforts are selecting for parasite strains that are able to persist at low densities without causing symptoms. This information is crucial to understanding what factors sustain transmission from submicroscopic gametocyte carriers even as overall transmission wans. Finally, Aim 3 deploys a field-relevant novel diagnostic test that has the potential to reshape malaria elimination strategies. We hypothesize that the sensitivity of Gam-RDT, a lateral flow immunoassay that detects a newly discovered gametocyte marker in saliva, approximates the gametocyte density at which transmission occurs, making it a valuable field tool for identifying parasite carriers who make up the infectious reservoir. We will conduct our study in Bagamoyo, Tanzania, an area where, although malaria cases have decreased over the last decade, up to ~45% of schoolchildren continue to have asymptomatic parasitemia detectable by highly sensitive PCR. Our team of experienced clinical trialists, leading malaria entomologists, and international experts in malaria epidemiology and genetic diversity will establish the evidence base for how malaria transmission from submicroscopic gametocyte carriers occurs in the face of intensive control efforts that have successfully reduced the global malaria burden, but will likely not be enough to eradicate this age-old human parasite.